Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-philPLA88 Virion-Associated Peptidoglycan Hydrolase HydH5: Fusions, Deletions and Synergy with LysH5

ABSTRACT

Virion-associated peptidoglycan hydrolases have a potential as antimicrobial agents due to their ability to lyse Gram positive bacteria on contact. Full-length HydH5, a virion-associated peptidoglycan hydrolase from the  Staphylococcus aureus  bacteriophage vB_SauS-phi-IPLA88, and two truncated derivatives, containing only the CHAP domain, exhibited high lytic activity against live  S. aureus  cells. Three different fusion proteins were created and showed higher staphylolytic activity than the parental enzyme or its deletion construct. Parental and fusion proteins lysed  S. aureus  cells in zymograms, plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human  S. aureus, S. aureus  MRSA N315 strain, and human  Staphylococcus epidermidis  strains. HydH5 and its derivative fusions proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro suggesting that the two enzymes have distinct cut sites and thus may be more efficient in combination for the elimination of staphylococcal infections.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of polypeptides having antimicrobialactivity and the polynucleotides encoding them. The invention alsorelates to nucleic acid constructs, vectors, and host cells comprisingthe nucleic acid constructs. The invention more specifically relates toan antimicrobial phage-associated HydH5 peptidoglycan hydrolasepolypeptide, truncations of the HydH5 peptidoglycan hydrolasepolypeptide, and fusion polypeptides comprising the HydH5 peptidoglycanhydrolase and truncated HydH5 polypeptides. This invention also relatesto synergistic pathogen-specific compositions comprising the endolysinLysH5 together either with HydH5 or with fusion polypeptides comprisingHydH5. The invention further relates to compositions and methods ofmaking the polypeptides and methods of treatingstaphylococcal-associated diseases, including methicillin-resistantStaphylococcus aureus (MRSA).

2. Description of the Relevant Art

Staphylococcus aureus is a notorious pathogen that causes numerouspathologies including food poisoning, toxic shock syndrome,endocarditis, and skin and wound infections, (Lowy, F. D. 1998. N. Engl.J. Med. 339:520-532). The emergence of multidrug-resistant strains,especially methicillin-resistant S. aureus (MRSA) andvancomycin-resistant S. aureus (VRSA) is raising serious concerns due totheir high frequency in both nosocomial and community-acquired settings(Köck et al. 2010. Euro Surveill. 15(41):19688).

Recent results with phage therapy in animal models have driven muchinterest in phages and phage-encoded proteins to treat infections(O'Flaherty et al. 2009. FEMS Microbiol. Rev. 33(4):801-819; Fenton etal. 2010. Bioeng. Bugs. 1(1):9-16). These studies clearly show theefficacy of phages and lysins in killing human pathogenic bacteria inanimal models (Matsuzaki et al. 2005. J. Infect. Chemotherapy11:211-219; Fischetti, V. A. 2010. Int. J. Med. Microbiol.300(6):357-362). Specifically, several assays have been performedagainst S. aureus bacteremia. The intraperitoneal administration ofphages phiMR11 and phiMR25 rescued mice inoculated with a lethal dose ofS. aureus (Matsuzaki et al. 2003. J. Infect. Dis. 187(4):613-624;Hoshiba et al. 2010. Arch. Virol. 155(4):545-552). Moreover, in a rabbitmodel of wound infection caused by S. aureus, phages prevented abscessformation (Wills et al. 2005. Antimicrob. Agents Chemother.49(3):1220-1221). Phage lytic proteins also showed successful results.The intraperitoneal administration of the endolysin MV-L from phagephiMR11 protected mice against S. aureus MRSA septic death (Rashel etal. 2007. J. Infect. Dis. 196(8):1237-1247). In another animal model,bacteremia in unprotected mice reached colony counts of ˜10⁷ cfu/mlwithin 3.5 h after challenge, whereas the administration of the lyticenzyme LysGH15 1 h after MRSA injection was sufficient to protect micewith the mean colony count being less than 10⁴ cfu/ml (Gu et al. 2011.J. Clin. Microbiol. 49(1):111-117). Furthermore, the activity of phagelytic proteins may be increased by using them in combination with otherantimicrobials. Both in vitro (Becker et al. 2008. FEMS Microbiol. Lett.287(2):185-191; Daniel et al. 2010. Antimicrob. Agents Chemother.54(4):1603-1612; Garcia et al. 2010. Int. J. Food Microbiol. 141(3):151-155) and in vivo synergy (Daniel et al., supra) between phageendolysin constructs and antibiotics or bacteriocins against S. aureushave been reported.

Bacterial cell walls of both Gram-positive and Gram-negative bacteriaare composed of peptidoglycan, a complex molecule with a sugar backboneof alternating N-acetylglucosamine and N-acetyl muramic acid residuescross-linked with peptide bridges. Peptidoglycan prevents osmotic lysisof cell protoplast and confers rigidity and shape on cells.Peptidoglycan hydrolases are essential for modifying the peptidoglycanto allow the cell to grow and divide (Vollmer et al. 2008. FEMSMicrobiol. Rev. 32(2):287-306). There are three major peptidoglycanhydrolase activities, namely, (i) glycosidase, (ii) amidase, and (iii)endopeptidase activities. Most peptidoglycan hydrolases are composed ofa C-terminal cell wall-binding (CWB) domain and a N-terminal catalyticdomain(s) (Fischetti, V. A. 2005. Trends in Microbiol. 13:491-496).Bacteriophages also encode peptidoglycan hydrolases (Hermoso et al.2007. Curr. Opin. Microbiol. 10(5):461-472) that play essential roles inthe phage life cycle to allow both entry (virion-associatedpeptidoglycan hydrolases) and release (endolysins) of the mature phageparticles.

In addition to endolysins, other phage encoded proteins(virion-associated peptidoglycan hydrolases) have a potential asantimicrobials. Some bacteriophage virions harbor virion-associatedpeptidoglycan hydrolases that facilitate the entry of phage DNA acrossthe bacterial cell envelope during infection (Moak and Molineux. 2004.Mol. Microbiol. 51:1169-1183). They are also responsible for the “lysisfrom without”, a phenomenon caused by some phages when adsorbed onto thehost cell at very high numbers (Delbrück, M. 1940. J. Gen. Physiol.23(5):643-660). This type of peptidoglycan hydrolase activity has beendescribed from a variety of different phage particles infecting S.aureus (Moak and Molineux, supra; Rashel et al. 2008. FEMS Microbiol.Lett. 284:9-16; Takac and Blasi. 2005. Antimicrob. Agents Chemother.49(7):2934-2940), Lactococcus lactis (Kenny et al. 2004. J. Bacteriol.186:3480-3491), E. coli (Molineux, I. J. 2001. Mol. Microbiol. 40:1-8;Kanamaru et al. 2002. Nature 415:553-557), and Salmonella (Steinbacheret al. 1997. J. Mol. Biol. 267:865-880).

Recently, a peptidoglycan hydrolase (HydH5) encoded by phagevB_SauS-philPLA88 (philPLA88) has been identified and characterized(Rodríguez et al. 2011, BMC Microbiol. 11: 138). HydH5 has a N-terminalCHAP (cysteine, histidine-dependent amidohydrolase/peptidase) lyticdomain and a C-terminal LYZ2 (lysozyme subfamily 2) lytic domain. HydH5does not have a recognized cell wall binding domain. The full-length 634amino acid HydH5 and truncated version harboring just one lytic domain(and 6×His-tag) have been overproduced in E. coli. The nickelchromatography purified proteins are able to kill viable S. aureuscells. HydH5 is highly thermostable since it showed antimicrobialactivity after heat treatment (100° C., 5 min) (Rodríguez et al.,supra).

Lysostaphin is a bacteriocin secreted by S. simulans that lyses S.aureus (Browder et al. 1965. Biochem. Biophys. Res. Commun. 19: 389).The endopeptidase activity is specific to the glycyl-glycyl bonds of thestaphylococcal peptidoglycan inter-peptide bridge. It is known thatlysostaphin can kill planktonic S. aureus (Walencka et al. 2005. Pol. J.Microbiol. 54: 191-200; Wu et al. 2003. Antimicrob. Agents Chemother.47: 3407-3414), as well as MRSA (Dajcs et al. 2000. Am. J. Ophthalmol.130: 544), vancomycin-intermediate S. aureus (Patron et al. 1999.Antimicrob. Agents Chemother. 43:1754-1755), and otherantibiotic-resistant strains of S. aureus (Peterson et al. 1978. J.Clin. Invest. 61: 597-609). Lysostaphin can also kill S. aureus growingin a biofilm (Walencka, supra; Wu, supra), and it exhibits limitedactivity against coagulase-negative staphylococci (Cisani et al. 1982.Antimicrob. Agents Chemother. 21: 531-535); McCormick et al. 2006. Curr.Eye Res. 31: 225-230).

The endolysin LysH5 is encoded by philPLA88 phage and has three putativedomains: a cysteine, histidine-dependent amidohydrolases/peptidase(CHAP) domain, an amidase-2 domain, and a C-terminal SH3b cellwall-binding (CWB) domain. LysH5 is able to inhibit the S. aureus growthin milk (Obeso et al. 2008. Int. J. Food Microbiol. 128(2):212-218) andshowed a synergistic antimicrobial effect with the bacteriocin nisin(Garcia et al. 2010, supra).

Antibiotic resistance in combination with other important virulencedeterminants, such as surface-located binding proteins to facilitateadhesion to host tissue, as well as many mechanisms to evade attack byhuman host defenses makes S. aureus a threatening pathogen (Otto, M.2010. Ann. Rev. Microbiol. 64:143-162). Novel therapeutic agentsspecific for staphylococcal species, including methicillin-resistantStaphylococcus aureus (MRSA), are sorely needed to counter the rise ofdrug resistant pathogenic bacteria.

SUMMARY OF THE INVENTION

We have discovered that the nucleic acid encoding the vB_SauS-philPLA88virion-associated peptidoglycan hydrolase HydH5, a protein whichspecifically attacks the peptidoglycan cell wall of untreated livestaphylococci can be truncated, that truncations encoding the CHAP(cysteine, histidine-dependent amidohydrolase/peptidase) lytic domain ofthe HydH5 peptidoglycan hydrolase result in polypeptides capable ofexolysis, i.e., “lysis from without” lytic activity, and thattruncations of HydH5 and fusion polypeptides comprising HydH5 ortruncations of HydH5 can be used as an antimicrobial treatment forStaphylococcal-induced infection and diseases, including infection anddisease caused by multidrug-resistant strains.

In accordance with this discovery, it is an object of the invention toprovide nucleic acid molecules encoding the truncated HydH5 polypeptidesand fusion proteins comprising the HydH5 peptidoglycan hydrolasepolypeptide and truncations of HydH5.

It is also an object of the invention to provide an antimicrobialtruncated HydH5 peptidoglycan hydrolase polypeptide which is functionalin that it comprises the CHAP domain and retains its properties forexolysis of the peptidoglycan cell wall of staphylococcal bacteria.

It is a further object of the invention to provide polynucleotidesencoding antimicrobial fusion proteins formed from a nucleic acidencoding a non-truncated HydH5 peptidoglycan hydrolase or encoding atruncated HydH5, i.e., a functioning CHAP domain, in combination with anucleic acid encoding lysostaphin or one or more of the SH3 cellwall-binding domain(s) of native lysostaphin.

It is a further object of the invention to provide antimicrobial fusionproteins comprising either a non-truncated HydH5 peptidoglycan hydrolaseor a functioning HydH5 CHAP domain from truncated HydH5 in combinationwith either lysostaphin or one or more of the SH3b cell wall-binding(CWB) domain(s) of native lysostaphin, i.e., HydH5 peptidoglycanhydrolase-lysostaphin, HydH5 peptidoglycan hydrolase-SH3b, andHydH5CHAP-SH3b.

It is another object of the invention to provide a method of usingantimicrobial fusion proteins comprising the CHAP domain from truncatedHydH5 peptidoglycan hydrolase in combination with lysostaphin or a SH3bcell wall-binding domain from lysostaphin to enhance the exolysis ofStaphylococcus strains and also extend the targets that can be “lysedfrom without” to additional bovine and human strains of Staphylococcusover and above the lysis observed with functional native HydH5peptidoglycan hydrolase comprising the native N-terminal CHAP domain incombination with the native C-terminal LYZ2 (lysozyme subfamily 2) lyticdomain.

An added object of the invention is to provide a nucleic acid sequenceencoding an antimicrobial HydH5 fusion polypeptide comprising thenucleic acid encoding the HydH5 peptidoglycan hydrolase or the truncatedHydH5 peptidoglycan hydrolase, i.e., the CHAP domain, in combinationwith nucleic acid encoding lysostaphin or a SH3b cell wall-bindingdomain from lysostaphin according to the invention as an encodingsequence which allows disease resistance to be imparted to the organism.It is well understood that this sequence can also be used in combinationwith another sequence, or sequences, encoding one or more diseaseresistant properties.

An additional object of the invention is to provide nucleic acidconstructs, vectors, and host cells comprising the nucleic acidconstructs encoding the fusion polypeptides of the invention.

An added object of the invention is to provide compositions useful forthe treatment of disease caused by the Staphylococcus strains for whichthe fusion proteins of the invention are specific and effective.

Another object of the invention is to provide compositions comprisingthe LysH5 endolysin together with HydH5 peptidoglycan hydrolase or withan HydH5-derived fusion protein selected from HydH5 peptidoglycanhydrolase-lysostaphin, HydH5 peptidoglycan hydrolase-SH3b, orHydH5CHAP-SH3b for treatment of diseases and infections caused byStaphylococcus strains where the composition comprises LysH5 and aparticular HydH5 fusion protein each in amounts that are ineffectivealone but act synergistically together to effectively treat saiddiseases and infections.

Also part of this invention is a kit, comprising a composition fortreatment of disease caused by the Staphylococcus strains for which theCHAP domain of the truncated HydH5 peptidoglycan hydrolase and fusionscomprising the truncated HydH5 peptidoglycan hydrolase CHAP domain arespecific and effective.

Other objects and advantages of this invention will become readilyapparent from the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of HydH5 deletion and fusionconstructs. The numbers indicate the initial and final amino acids inthe domains as determined by Pfam domain database. Grey box: CHAPdomain; horizontal stripes: LYZ2 domain; diagonal stripes=SH3b domain;large black box: endopeptidase domain; small black box: 6×His-tag.

FIGS. 2A, 2B, and 2C depict the antimicrobial activity of the HydH5,deletion and fusion constructs. FIG. 2A shows the SDS-PAGE and zymogramanalyses of 5 μg nickel affinity-purified proteins. Lane M: Standardmolecular mass marker in kDa (Prestained SDS-PAGE Standards, broadrange; BioRad Laboratories); lane 1, CHAP (19.6 kDa); 2, HydH5 (73.6kDa); 3, HydH5SH3b (85.1 kDa); 4, HydH5Lyso (100.8 kDa); 5, CHAPSH3b(30.4 kDa); 6, Lysostaphin (28.1 kDa). FIG. 2B shows a plate lysis assayof HydH5 and derived proteins using mid-log phase growing cells of S.aureus Sa9 and MRSA strain N315. FIG. 2C shows turbidity reduction assayresults using 1 μM of each protein. Specific activity is expressed asΔOD_(600 nm) min⁻¹ μM⁻¹. Error bars are the means±standard deviations ofthree independent assays.

FIG. 3 shows the synergistic effect in checkerboard assay betweenendolysin LysH5 and HydH5 fusion constructs. Minimum Lytic Concentration(MLC) of each protein in the presence of subinhibitory concentrations ofendolysin LysH5 is indicated. A (HydH5, 56.7 μg/ml to 0.06 μg/ml, ΣFIC0.065±0.001), B (HydH5SH3b, 13.5 μg/ml to 0.01 μg/ml, ΣFIC 0.130±0.000),C (HydH5Lyso, 25.1 μg/ml to 0.02 μg/ml, ΣFIC 0.049±0.023), and D(CHAPSH3b, 17.34 μg/ml to 0.03 μg/ml, ΣFIC 0.063±0.022).

DETAILED DESCRIPTION OF THE INVENTION

In order to enhance the native antimicrobial activity of HydH5peptidoglycan hydrolase against target staphylococcal species andstrains, in this work we have fused HydH5 to both full length andtruncated fragments of lysostaphin, a staphylolytic bacteriocin producedby Staphylococcus simulans. A chimeric protein made up of complete HydH5and lysostaphin fused in a head to tail configuration was created.Similarly, both the complete HydH5 protein and just the HydH5 CHAPdomain were fused to the SH3b CWB domain of lysostaphin in two separateconstructs. The enzybiotic activity of all these fusion proteins towardslive S. aureus and numerous non-staphylococcal bacteria was assessed.The list of pathogen species effectively lysed by proteins encoded bythe constructs of invention has been extended to include both bovine andhuman staphylococcal strains. Finally, we have also evaluated possiblesynergistic effects between HydH5 and the endolysin LysH5 with thepurpose to explore new enzybiotic-based strategies to fight S. aureusinfections.

In our previous work (Rodriguez et al. 2011, supra), we demonstrated theantimicrobial activity of HydH5 peptidoglycan hydrolase against S.aureus Sa9 strain. With the goal to increase the HydH5 lytic activity weperformed deletion analysis and created fusion proteins with the mostactive deletion constructs. Through deletion analysis we had identifiedin HydH5 the protein domains that are essential for its activity andshowed that both CHAP and LYZ2 domains possess lytic activity against S.aureus cells (Rodriguez et al. 2011, supra). Previous to this workseveral reports have shown that endolysins can be truncated and someindividual catalytic domains retain lytic activity with some of themshowing higher specific activity than the full length protein (Cheng etal. 2007. Appl. Microbiol. Biotechnol. 74(6):1284-1291; Horgan et al.2009. Appl. Environ. Microbiol. 75(3):872-874; Donovan et al. 2006.Appl. Environ. Microbiol. 72:5108-5112; Becker et al. 2008, supra).However, as was previously shown, some endolysin catalytic domains arealmost inactive, such as the glycosidase domains from lambdaSa2 prophageendolysin (Donovan and Foster-Frey. 2008. FEMS Microbiol. Lett.287(1):22-33), or the B30 endolysin (Donovan et al. 2006, supra) and theamidase domain from LysK (Becker et al. 2008, supra). Although theremight be protein stability or aberrant protein folding influencing ourresults, the HydH5 CHAP domain construct has approximately 1.4-foldlower specific activity compared to the activity of the full lengthHydH5, suggesting that LYZ2 domain also contributes to the completeprotein activity.

We and others have shown previously that virion-associated peptidoglycanhydrolases lacking a defined CWB domain can also bind to the cellsurface (Rashel et al. 2008, supra; Rodriguez et al. 2011, supra). KnownCWB domains have been shown for some peptidoglycan hydrolases to benecessary for accurate cell wall recognition and subsequent lyticactivity such as lysostaphin (Baba and Schneewind. 1996. EMBO J.15(18):4789-4797), ALE-1 (Lu et al. 2006. J. Biol. Chem.281(1):549-558), L. monocytogenes endolysins Ply118 and Ply500 (Loessneret al. 2002. Mol. Microbiol. 44(2):335-349) and S. pneumoniae CPL-1(Pérez-Dorado et al. 2007. J. Biol. Chem. 282(34):24990-24999). However,there are numerous reports of C-terminally deleted lysin constructs withdeleted CWB domains where the N-terminal lytic domain maintains itsactivity in the absence of its CWB domain (Cheng et al., supra; Sass andBierbaum. 2007. Appl. Environ. Microbiol. 73(1):347-352; Horgan et al.,supra; Donovan et al. 2006, supra; Becker et al. 2008, supra). Thus adefined CWB domain is not a necessity for full lytic activity, and thisbinding does not rule out the possibility that the inter-lytic domainregion might harbour a heretofore unidentified CWB domain as suggestedpreviously for the B30 endolysin (Donovan et al. 2006, supra;lysostaphin/B30 fusion).

Our hypothesis to justify the addition of a CWB domain considers thefact that a phage structural protein, HydH5, is likely brought intoclose proximity of the cell wall and the peptidoglycan substrate by theintact virion, meaning that a CWB domain is likely not a strictrequirement. However, as a soluble single protein antimicrobial, thescenario is quite different and the addition of a CWB domain mightimprove its lytic activity. Our SDS PAGE and zymogram results show thatfor all constructs, we are working with >95% pure proteins. In theHydH5-SH3b construct, the lysostaphin SH3b domain was fused tofull-length HydH5 increasing the lytic activity 1.7-fold, suggestingthat this CWB domain helps the lytic domain to degrade the peptidoglycansubstrate. A similar result was obtained when the native Cpl-7 cell wallbinding domains of the streptococcal LambdaSa2 endolysin was replaced bystaphylococcal SH3b domain from lysostaphin or LysK resulting in a 5×increase in staphylolytic activity (Becker et al. 2009. FEMS Microbiol.Lett. 294(1):52-60). In the HydH5CHAP-SH3b construct, the lysostaphinSH3b domain was fused to the CHAP domain of HydH5 resulting in a4.8-fold increase in the lytic activity of the CHAP domain aloneconstruct (CHAP) and a 3.3-fold increase compared to full length HydH5.This result indicates the importance of the SH3b CWB domain inrecognizing the bacteria cell wall, taking into account that only onecatalytic domain (CHAP) fused to the SH3b domain was sufficient toobtain the highest specific activity of the various constructs.

Moreover, addition of the full length lysostaphin to HydH5(HydH5-lysostaphin) also achieved greater activity levels than theparental protein (HydH5). This result further indicates the modularnature of these protein domains and that the addition of new catalyticdomains to HydH5 (i.e., the glycyl-glycine endopeptidase of lysostaphin)can also improve its lytic activity. The three different catalyticdomains are believed to each attack a different peptidoglycan bond. Thepresence of multiple unique domains theoretically decreases thelikelihood of bacterial resistance development (Fischetti, V. A. 2005,supra). The increased activity also suggests that each domain ismodular, as expected (Garcia et al. 1990. Gene 86(1):81-88), andalthough the activity of the final construct is not the sum of parentallytic activities, each domain likely achieves a near-nativeconformation.

HydH5 and its derivatives fusions were assessed for their ability tolyse a number of staphylococcal and non-staphylococcal strains. Noprevious data have been reported regarding the lytic spectrum of S.aureus virion-associated peptidoglycan hydrolases. All proteins wereactive against staphylococcal strains but no other genus was lysed atdetectable levels. Our results indicate a similar lytic spectrum forboth HydH5 and its derivative fusions. These results are in agreementwith S. aureus endolysins described to date that demonstrate a lyticspectrum limited to staphylococci. However, a different level of thelytic activity was observed for each species/strain. In general, S.aureus strains were more sensitive than S. epidermidis strains. Withinthe S. aureus strains, we observed that bovine strains were moresensitive than clinical strains. This could be due to the shared originof these strains and the phage from which HydH5 originated; both share asource from a dairy environment. A similar result was previouslyobserved for endolysin LysH5 (Obeso et al., supra). Although we do notyet have direct biochemical proof that all three lytic domains arefunctional in the HydH5-lysostaphin construct, the reduced activityagainst S. epidermidis compared to S. aureus is consistent with thelytic range of lysostaphin and suggests that the lysostaphin domain isfunctional in this construct, given that lysostaphin is known to have areduced activity against S. epidermidis compared to S. aureus (Zygmuntet al. 1968. Appl. Microbiol. 16(8):1168-1173).

Synergistic interactions between antimicrobial compounds have thepotential to be exploited in order to increase effectiveness against thetarget bacteria, thereby reducing the dose required of each whiledecreasing the likelihood of antimicrobial-resistance. The combinationof the two lytic bacteriophage enzymes HydH5 and LysH5 appears to havesynergistic activity on S. aureus Sa9 strain. The HydH5-derived fusionsalso showed this positive interaction. The basis of synergy betweenpeptidoglycan hydrolases might be explained by the hydrolytic activityof one enzyme loosening the peptidoglycan structure, thus facilitatingbetter access of the second enzyme to its target. It is encouraging thatthe strongest in vitro synergy was observed between LysH5 andHydH5-lysostaphin, suggesting that this combination might be protectivein vivo. However, in the absence of biochemical data to indicate whichdomains are active in the fusion and the parental LysH5, we cannotidentify the particular domains that are responsible for the observedantimicrobial synergy. However, these results are in agreement withprevious studies indicating synergy between Pal and Cpl-1 lysins againstS. pneumoniae (Loeffler and Fischetti. 2003. Antimicrob. AgentsChemother. 47(1):375-377), and the synergy between endolysin LysK andthe bacteriocin lysostaphin against S. aureus. A 33% reduction in LysKconcentration was obtained in the presence of lysostaphin (Becker et al.2008, supra). Another bacteriocin, nisin, enhanced 8-fold the lyticactivity of LysH5 on S. aureus cell suspensions (García et al. 2010,supra). Comparable results were obtained by combination with antibiotics(Manoharadas et al. 2009. J. Biotechnol. 139:118-123; Daniel et al.,supra). However, the effect of a second antimicrobial compound onreducing the development of resistant staphylococcal strains is also aconsideration.

In summary, we report the development of novel chimeric peptidoglycanhydrolases with improved lytic activity against S. aureus and S.epidermidis, including MRSA N315 strain. The effectiveness of HydH5 andits derivative fusions when used in combination with LysH5, anotherdairy-derived protein were remarkable. We expect that these constructswill provide new weapons to combat multidrug-resistant S. aureusinfections in both dairy and clinical environments.

The present invention also relates to a chimeric gene (or expressioncassette) comprising an encoding sequence as well as heterologousregulatory elements in positions 5′ and 3′ which can function in a hostorganism, the encoding sequence comprising at least one nucleic acidsequence encoding an isolated truncated HydH5 peptidoglycan hydrolaserelated protein (truncation or fusion) as defined above. By hostorganism there is to be understood any single-celled or lower or highernon-human multi-celled organism into which HydH5 peptidoglycan hydrolasegene according to the invention can be introduced. The regulatoryelements required for expressing the nucleic acid sequence encoding atruncated HydH5 peptidoglycan hydrolase are well known to those skilledin the art and depend on the host organism. The means and methods foridentifying and choosing the regulatory elements are well known to thoseskilled in the art and widely described in the literature.

The present invention also relates to a cloning and/or expression vectorfor transforming a host organism containing at least the truncated HydH5peptidoglycan hydrolase gene as defined hereinabove. This vectorcomprises, in addition, to the above truncated HydH5 peptidoglycanhydrolase gene, at least one replication origin. This vector can beconstituted by a plasmid, a cosmid, a bacteriophage or a virus which istransformed by introducing the chimeric gene according to the invention.Such transformation vectors according to the host organism to betransformed are well known to those skilled in the art and widelydescribed in the literature.

A further subject of the invention is a process for the transformationof host organisms, by integrating a least one nucleic acid sequence orchimeric gene as defined hereinabove, which transformation may becarried out by any suitable known means which have been widely describedin the specialist literature and in particular in the references citedin the present application, more particularly by the vector according tothe invention.

According to the present invention, the terms “nucleic acid molecule”,“nucleic acid sequence”, “polynucleotide”, “polynucleotide sequence”,“nucleic acid fragment”, “isolated nucleic acid fragment” are usedinterchangeably herein. These terms encompass nucleotide sequences andthe like. A polynucleotide may be a polymer of RNA or DNA that issingle- or double-stranded and that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. This will also includea DNA sequence for which the codons encoding the HydH5 peptidoglycanhydrolase according to the invention will have been optimized accordingto the host organism in which it will be expressed, these optimizationmethods being well known to those skilled in the art.

The term “isolated” polynucleotide refers to a polynucleotide that issubstantially free from other nucleic acid sequences, such as otherchromosomal and extrachromosomal DNA and RNA, that normally accompany orinteract with it as found in its naturally occurring environment.However, isolated polynucleotides may contain polynucleotide sequenceswhich may have originally existed as extrachromosomal DNA but exist as anucleotide insertion within the isolated polynucleotide. Isolatedpolynucleotides may be purified from a host cell in which they occur.Conventional nucleic acid methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term embraces cDNA, recombinantpolynucleotides and chemically synthesized polynucleotides.

The term “transformation” refers to a permanent or transient geneticchange induced in a cell following the incorporation of new DNA (i.e.DNA exogenous to the cell). Where the cell is a mammalian cell, apermanent genetic change is generally achieved by introduction of theDNA into the genome of the cell. When the cell is a bacterial cell, theterm usually refers to an extrachromosomal, self-replicating vectorwhich harbors a selectable antibiotic resistance. Thus, isolatedpolynucleotides of the present invention can be incorporated intorecombinant constructs, typically DNA constructs, capable ofintroduction into and replication in a host cell. Such a construct canbe a vector that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell.

The term “construct” refers to a recombinant nucleic acid, generallyrecombinant DNA, that has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences. A “construct” or“chimeric gene construct” refers to a nucleic acid sequence encoding aprotein, operably linked to a promoter and/or other regulatorysequences.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter) or a DNA sequenceand a regulatory sequence(s) are connected in such a way as to permitgene expression when the appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the regulatory sequence(s).

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence that can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter.

The term “cDNA” refers to all nucleic acids that share the arrangementof sequence elements found in native mature mRNA species, where sequenceelements are exons and 3′ and 5′ non-coding regions. Normally mRNAspecies have contiguous exons, with the intervening introns removed bynuclear RNA splicing, to create a continuous open reading frame encodingthe protein. “cDNA” refers to a DNA that is complementary to and derivedfrom an mRNA template.

The term “genomic sequence” refers to a sequence having non-contiguousopen reading frames, where introns interrupt the protein coding regions.It may further include the 3′ and 5′ untranslated regions found in themature mRNA. It may further include specific transcriptional andtranslational regulatory sequences, such as promoters, enhancers, etc.,including about 1 kb, but possibly more, of flanking genomic DNA ateither the 5′ or 3′ end of the transcribed region. The genomic DNA maybe isolated as a fragment of 100 kbp or smaller; and substantially freeof flanking chromosomal sequence.

As used herein, “recombinant” refers to a nucleic acid molecule whichhas been obtained by manipulation of genetic material using restrictionenzymes, ligases, and similar genetic engineering techniques asdescribed by, for example, Sambrook et al. 1989. Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. or DNA Cloning: A Practical Approach, Vol. Iand II (Ed. D. N. Glover), IRL Press, Oxford, 1985. “Recombinant,” asused herein, does not refer to naturally occurring geneticrecombinations.

As used herein, the term “chimeric” refers to two or more DNA moleculeswhich are derived from different sources, strains, or species, which donot recombine under natural conditions, or to two or more DNA moleculesfrom the same species, which are linked in a manner that does not occurin the native genome.

As used herein, the terms “encoding”, “coding”, or “encoded” when usedin the context of a specified nucleic acid mean that the nucleic acidcomprises the requisite information to guide translation of thenucleotide sequence into a specified protein. The information by which aprotein is encoded is specified by the use of codons. A nucleic acidencoding a protein may comprise non-translated sequences (e.g., introns)within translated regions of the nucleic acid or may lack suchintervening non-translated sequences (e.g., as in cDNA).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide. Each protein or polypeptide has a uniquefunction.

The invention includes functional fragments of the HydH5 peptidoglycanhydrolase polypeptide and functional fusion polypeptides encompassing afunctional HydH5 peptidoglycan hydrolase and functional fragmentsthereof, as well as mutants and variants having the same biologicalfunction or activity. As used herein, the terms “functional fragment”,“mutant” and “variant” refers to a polypeptide which possessesbiological function or activity identified through a defined functionalassay and associated with a particular biologic, morphologic, orphenotypic alteration in the cell. The term “functional fragments ofHydH5 peptidoglycan hydrolase” refers to all fragments of HydH5peptidoglycan hydrolase that retain HydH5 peptidoglycan hydrolaseactivity and function to lyse staphylococcal bacteria.

Modifications of the HydH5 peptidoglycan hydrolase primary amino acidsequence may result in further mutant or variant proteins havingsubstantially equivalent activity to the HydH5 peptidoglycan hydrolasepolypeptides described herein. Such modifications may be deliberate, asby site-directed mutagenesis, or may occur by spontaneous changes inamino acid sequences where these changes produce modified polypeptideshaving substantially equivalent activity to the HydH5 peptidoglycanhydrolase polypeptide. Any polypeptides produced by minor modificationsof the HydH5 peptidoglycan hydrolase primary amino acid sequence areincluded herein as long as the biological activity of HydH5peptidoglycan hydrolase is present; e.g., having a role in pathwaysleading to lysis of staphylococcal bacteria.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of nucleotides thatdo not substantially affect the functional properties of the resultingtranscript. It is therefore understood that the invention encompassesmore than the specific exemplary nucleotide or amino acid sequences andincludes functional equivalents thereof. Alterations in a nucleic acidfragment that result in the production of a chemically equivalent aminoacid at a given site, but do not affect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. A method of selecting an isolated polynucleotide that affectsthe level of expression of a polypeptide in a host cell may comprise thesteps of: constructing an isolated polynucleotide of the presentinvention or an isolated chimeric gene of the present invention;introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; measuring the level of a polypeptide in the host cellcontaining the isolated polynucleotide; and comparing the level of apolypeptide in the host cell containing the isolated polynucleotide withthe level of a polypeptide in a host cell that does not contain theisolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (1985.Nucleic Acid Hybridization, Hames and Higgins, Eds., IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. An indication thatnucleotide sequences are substantially identical is if two moleculeshybridize to each other under stringent conditions. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C., depending upon the desired degree ofstringency as otherwise qualified herein. Thus, isolated sequences thatencode a HydH5 peptidoglycan hydrolase polypeptide and which hybridizeunder stringent conditions to the HydH5 peptidoglycan hydrolasesequences disclosed herein, or to fragments thereof, are encompassed bythe present invention.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Methods of alignment of sequences for comparison are well known inthe art. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988. CABIOS 4:11-17), the local homology algorithmof Smith et al. (1981. Adv. Appl. Math. 2:482); the homology alignmentalgorithm of Needleman and Wunsch (1970. J. Mol. Biol. 48:443-453); thesearch-for-similarity-method of Pearson and Lipman (1988. Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990.Proc. Natl. Acad. Sci. USA 87:2264), modified as in Karlin and Altschul(1993. Proc. Natl. Acad. Sci. USA 90:5873-5877).

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins, it is recognizedthat residue positions which are not identical often differ byconservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 80% sequenceidentity, preferably at least 85%, more preferably at least 90%, mostpreferably at least 95% sequence identity compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 80%, preferably atleast 85%, more preferably at least 90%, and most preferably at least95%. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman et al. (1970. J. Mol. Biol. 48:443).

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST. In general, a sequence of ten ormore contiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification and isolation. Inaddition, short oligonucleotides of 12 or more nucleotides may be use asamplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises a nucleotide sequence thatwill afford specific identification and/or isolation of a nucleic acidfragment comprising the sequence. The instant specification teachesamino acid and nucleotide sequences encoding polypeptides that comprisea particular plant protein. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Thus, such a portion represents a “substantial portion” andcan be used to establish “substantial identity”, i.e., sequence identityof at least 80%, compared to the reference sequence. Accordingly, theinstant invention comprises the complete sequences as reported in theaccompanying Sequence Listing, as well as substantial portions at thosesequences as defined above.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.By “fragment” a portion of the nucleotide sequence or a portion of theamino acid sequence and hence protein encoded thereby is intended.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence haveHydH5 peptidoglycan hydrolase-like activity. Alternatively, fragments ofa nucleotide sequence that are useful as hybridization probes may notencode fragment proteins retaining biological activity.

By “variants” substantially similar sequences are intended. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the HydH5 peptidoglycan hydrolase polypeptidesof the invention. Naturally occurring allelic variants such as these canbe identified with the use of well-known molecular biology techniques,as, for example, with polymerase chain reaction (PCR), a technique usedfor the amplification of specific DNA segments. Generally, variants of aparticular nucleotide sequence of the invention will have generally atleast about 90%, preferably at least about 95% and more preferably atleast about 98% sequence identity to that particular nucleotide sequenceas determined by sequence alignment programs described elsewhere herein.

By “variant protein” a protein derived from the native protein bydeletion (so-called truncation) or addition of one or more amino acidsto the N-terminal and/or C-terminal end of the native protein; deletionor addition of one or more amino acids at one or more sites in thenative protein; or substitution of one or more amino acids at one ormore sites in the native protein is intended. Variant proteinsencompassed by the present invention are biologically active, that isthey possess the desired biological activity, that is, HydH5peptidoglycan hydrolase activity as described herein. Such variants mayresult from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of a native HydH5peptidoglycan hydrolase protein of the invention will have at leastabout 90%, preferably at least about 95%, and more preferably at leastabout 98% sequence identity to the amino acid sequence for the nativeprotein as determined by sequence alignment programs described elsewhereherein. A biologically active variant of a protein of the invention maydiffer from that protein by as few as 1-15 amino acid residues, or even1 amino acid residue.

The polypeptides of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Novel proteins having properties of interest may be createdby combining elements and fragments of proteins of the presentinvention, as well as with other proteins. Methods for suchmanipulations are generally known in the art. Thus, the genes andnucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant forms. Likewise, the proteins ofthe invention encompass naturally occurring proteins as well asvariations and modified forms thereof. Such variants will continue topossess the desired HydH5 peptidoglycan hydrolase activity. Obviously,the mutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays where the effects of HydH5peptidoglycan hydrolase protein can be observed.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein.

The staphylococcal control compositions of the invention comprise theantimicrobial composition of the invention dissolved or suspended in anaqueous carrier or medium. The composition may further generallycomprise an acidulant or admixture, a rheology modifier or admixture, afilm-forming agent or admixture, a buffer system, a hydrotrope oradmixture, an emollient or admixture, a surfactant or surfactantadmixture, a chromophore or colorant, and optional adjuvants. Thepreferred compositions of this invention comprise ingredients which aregenerally regarded as safe, and are not of themselves or in admixtureincompatible with milk or milk by-products or human and veterinaryapplications. Likewise, ingredients may be selected for any givencomposition which are cooperative in their combined effects whetherincorporated for antimicrobial efficacy, physical integrity of theformulation or to facilitate healing and health in medical andveterinary applications, including for example in the case of mastitis,healing and health of the teat. or other human or animal body part.Generally, the composition comprises a carrier which functions to dilutethe active ingredients and facilitates stability and application to theintended surface. The carrier is generally an aqueous medium such aswater, or an organic liquid such as an oil, a surfactant, an alcohol, anester, an ether, or an organic or aqueous mixture of any of these. Wateris preferred as a carrier or diluent in compositions of this inventionbecause of its universal availability and unquestionable economicadvantages over other liquid diluents.

Avoiding the generalized use of broad range antimicrobials and usinghighly specific antimicrobials for just the target organisms involvedshould help reduce the ever-increasing incidence of antibioticresistance.

EXAMPLES

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples, which are includedherein only to further illustrate the invention and are not intended tolimit the scope of the invention as defined by the claims.

Example 1 Bacterial Strains and Culture Conditions

Bacterial strains used to determine lytic spectrum of proteins arelisted in Table 1. S. aureus Sa9 was used as the indicator strain forlytic activity (Obeso et al., supra). The S. aureus bovine isolates werefrom in house stocks (IPLA-CSIC), the S. aureus clinical isolates by Dr.Suárez (University of Oviedo, Spain), the S. epidermidis by Dr. Delgado(IPLA-CSIC) and the S. aureus MRSA strain N315 by Prof. Gabi Bierbaum(University of Bonn, Germany). All Staphylococcus strains were grown inTSB broth (Tryptic Soy Broth, DIFCO, Franklin Lakes, N.J.) at 37° C.with shaking or in TSB plates containing 2% (w/v) bacteriological agar(TSA). Other bacteria strains belonging to several different genera suchas Bacillus, Streptococcus, Listeria, Enterococcus, Clostridium,Lactococcus, Leuconostoc and Lactobacillus, were insensitive to thelytic proteins (data not shown). Bacillus, Streptococcus, Listeria andEnterococcus strains were grown in 2×YT at 37° C. with shaking.Clostridium strain was grown in BHI (Brain Heart Infusion, Scharlau,Barcelona, Spain) at 30° C. under static conditions and anaerobiosis(Anaerocult A, Merck, Darmstadt, Germany). Lactococcus strain was grownin lactose-M17 (Scharlau, Barcelona, Spain) at 30° C. and static.Leuconostoc and Lactobacillus strains were grown in MRS (Scharlau,Barcelona, Spain) at 30° C. and static.

Example 2 Plasmid Constructs; DNA Manipulation

Bacteriophage vB_SauS-philPLA88 gene orf58 encoding HydH5 (Acc. NumberACJ64586) was codon-optimized (orf58-opt) based on the E. coli codonusage (Laird et al. 2005. Protein Expr. Purif. 39(2):237-246) andcommercially synthesized (Genescript, Piscataway, N.J., USA). Thenorf58-opt was synthesized with a 5′ Ndel and a 3′ Xhol restrictionenzyme sites and the resultant fragment was subcloned between the Ndeland Xhol sites in the multi cloning site of the inducible expressionvector pET21a (EMD Biosciences, San Diego, Calif.) which introduces aC-terminal 6×His-tag. All constructions in this work were created inthis vector and thus have two additional amino acids residues introducedat the C-terminus corresponding to Xhol site (Leu-Glu) followed by the6×His-tag. Truncated versions of HydH5 were constructed via PCRamplification by introducing cloning sites via the PCR primers: Xholsites (underlined) after aa codon 156 for CHAP156 domain (CHAP156R:5′-ACTGACTGCTCGAGTTT GTCCGGGTG-3′; SEQ ID NO: 1) or after aa codon 166for CHAP166 domain (CHAP166R: 5′-ATCGACTGCTCGAGTTTCGGCACCGG-3′; SEQ IDNO: 2) and a Ndel site before aa codon 475 for LYZ161 domain (LYZ161F:5′-ACTGACTGCATATGGTTAGCGTCTCC-3′; SEQ ID NO: 3) or before aa codon 465for LYZ171 domain (LYZ171F: 5′-ACTGACTGCATATGCAAATGCTGAAC-3′; SEQ ID NO:4). Forward primer for CHAP domains was pET21aBglll-F: 5′-CGTAGAGGATCGAGATCTCGATC-3′ (SEQ ID NO: 5) and reverse primer for LYZ2 domainswas pET21aStyl-R: 5′-CGT TTAGAGGCCCCAAGGGGTTATG-3′ (SEQ ID NO: 6). Thefull-length lysostaphin gene was amplified with PCR pairs Full LysoSall-F (5′-ATC ATCGTCGACGCTGCAACAC ATGAACATTCAGCAC-3′; SEQ ID NO: 7) andpET21aStyl-R. The lysostaphin fragment encoding the SH3b domain wasamplified with PCR primer pairs Lyso Sall144-F(5′-GGAAAAGCAGTCGACACAGTAACTCC-3′; SEQ ID NO: 8) and pET21aStyl-R. Theseamplified fragments have an N-terminal Sa/l restriction site(underlined) designed to allow in-frame gene fusions of full-lengthlysostaphin or its SH3b domain individually to the C-terminus of HydH5or its catalytic domain CHAP156. Vector constructs were performed in E.coli DH5α cells (Invitrogen, Carlsbad, Calif.) and induced in E. coliBL21 (DE3) (EMD Biosciences, San Diego, Calif.).

To determine whether both the CHAP and LYZ2 as single domains showedincreased lytic activity toward S. aureus Sa9 over those previouslyobtained (Rodriguez et al. 2011.) four deletion constructs weregenerated. A schematic representation of all constructs is presented inFIG. 1. In order to ensure proper folding, each construct encoded asingle lytic domain with an additional sequence of either 7 or 17 aminoacids surrounding the catalytic domain (CHAP156, CHAP166, LYZ161 andLYZ171). In addition, due to the lack of a known cell wall bindingdomain in HydH5, we proceeded to determine whether the addition of acell wall binding domain might increase the lytic activity of HydH5 andCHAP. Therefore, three different fusion proteins were created betweenlysostaphin and HydH5 (FIG. 1). Initially, the nucleic acid encoding thelysostaphin binding domain SH3b was fused to the nucleic acid encodingthe full-length HydH5 protein, resulting in a protein with two catalyticdomains and one cell wall binding domain, i.e., the protein HydH5-SH3b(SEQ ID NO: 10). A second construct was obtained by fusion of thenucleic acid encoding the CHAP156 domain to the nucleic acid encodingthe lysostaphin SH3b domain resulting in the protein HydH5CHAP-SH3b (SEQID NO: 12). Finally, the nucleic acid encoding the full-lengthlysostaphin and the full-length HydH5 were fused in order to obtain theprotein HydH5-Lysostaphin (SEQ ID NO: 14) with three catalytic domainsand a cell wall binding domain. All the fusion proteins could bedetected and purified with the exception of LYZ161 and LYZ171 whoseproducts could not be detected after nickel column purification of theE. coli cultures.

The nucleic acid molecules encoding the constructs HydH5-SH3b,HydH5CHAP-SH3b, and HydH5-Lysostaphin, comprise nucleotides encodingC-terminal Leu-Glu-His-His-His-His-His-His residues and are identifiedby SEQ ID NOs: 9, 11, and 13, respectively. The expressed proteins areHis-tagged with eight additional amino acid residues introduced at theC-terminus corresponding to the Xhol site (Leu-Glu) followed by six Hisresidues. The proteins encoded by these nucleic acid sequences areidentified by SEQ ID NOs: 10, 12 and 14, respectively. The constructsencoding CHAP156, CHAP166, LYZ161 and LYZ171 are identified by SEQ IDNOs: 15, 17, 19 and 21, respectively, and the resulting proteins, by SEQID NOs: 16, 18, 20 and 22, respectively.

Example 3 Protein Purification and Analysis

Protein purification was performed as previously described (Donovan andFoster-Frey, supra) with the following modifications: Exponentialgrowing cultures induced by IPTG (1 mM, final concentration) wereincubated at 10²C for 20 h. Then 500 ml pellets where sonicated for 5min using an automatic pulsing sonication (Bronson Sonifier; BronsonSonic Power Co., Danbury, Conn., USA). Protein purification was carriedout by NiNTA nickel column chromatography (Qiagen). Wash and elutionprofiles were empirically determined to be 20 ml of 10 mM imidazole, 40ml of 20 mM imidazole and elution with 1 ml of 250 mM imidazole inphosphate buffered saline (50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0) with 30%glycerol to prevent precipitation of the purified protein. Then allsamples were converted to HydH5 activity buffer (HEPES 50 mM, NDSB-2010.5 M, CaCl₂ 0.25 mM, MnCl₂ 0.25 mM, MgCl₂ 0.25 mM, TCEP 1 mM, NaCl 24mM, KCl 1 mM pH 7.5)(Rodriguez et al. 2011.) containing 30% glycerolusing pre-equilibrated Zeba Desalting Columns (Thermo Fisher Scientific,Rockford, Ill.).

Purity of HydH5 constructs were evaluated in a 15% (v/w) SDS-PAGE inTris-Glycine buffer at 150 V for 1.5 h using Criterion Precast gels(Bio-Rad, Inc., Hercules, Calif.). The proteins purified on the nickelcolumn retained the His tags, thus the His tags were present in thevarious assays. His tags can be removed by methods well known andpracticed in the art.

Example 4 Zymogram, Plate Lysis and Turbidity Reduction Assays

Due to reports that peptidoglycan hydrolase lytic activity is not alwaysquantitatively comparable between multiple assays although it usuallyagrees qualitatively (Kusuma and Kokai-Kun. 2005. Antimicrob. AgentsChemother. 49(8): 3256-3263), the lytic activities of the other proteins(HydH5, CHAP156, CHAP166, HydH5SH3b, CHAPSH3b and HydH5Lyso) weredetermined in three antimicrobial assays: zymogram, plate lysis andturbidity reduction assays. The turbidity reduction assay requires arobust enzyme in order to lyse large numbers of cells (startingconcentration is >10⁹ cells/ml), while the plate lysis assay can definerelative activity with much lower amounts of bacteria, approximately 10⁶cells.

Zymogram assays were performed as previously described (Rodriguez et al.2011, supra). SDS gels were stained via conventional Coomassie stainingand zymograms were soaked for 30 min in distilled water to remove SDSand then incubated at room temperature in water for 15 min to detectareas of clearing in the turbid gel where a lytic protein is localized.

As shown in FIG. 2A, all fusion proteins showed a single zone ofclearing in the zymogram consistent with the predicted molecular massand positions of the purified proteins in the SDS-PAGE. In addition, thetruncation constructs (CHAP156 and CHAP166) also had the ability to lyseS. aureus. The activity of both truncated constructs (CHAP156 andCHAP166) turned out to be the same regardless of the length of the lyticdomain. Therefore, only the results obtained with CHAP156 are shown.

Plate lysis was performed as previously described (Donovan andFoster-Frey, supra). Purified proteins for each construct were dilutedin HydH5 activity buffer. Ten microliters of a 4 μM solution and 1:2dilutions of each construct were spotted onto a freshly spread lawn ofS. aureus Sa9, S. aureus MRSA N315 and the strains of Table 1 that hadair dried for 30 min on tryptic soy agar plates. The spotted plates wereair dried for 10 min in a laminar flow hood, and incubated overnight ina 37° C. environment. A cleared spot in the lawn indicates lysis of thepathogen. Scoring of the cleared spots occurred within 20 hr of platingthe cells.

In the plate lysis assay (FIG. 2B) the full-length HydH5 showed clearingof S. aureus at ≧2 μM concentration while the HydH5CHAP domain requiredhigher concentrations (≧16 μM). All the fusion proteins showedsignificantly greater activity than the parental protein HydH5, evenagainst a methicillin-resistant S. aureus MRSA N315 strain. HydH5 hasclear staphylolytic activity at 4 μM in contrast with fusion proteinswhich shown lysis activity at much lower concentrations (1 μM). Fusionof SH3b to the CHAP domain (HydH5CHAP-SH3b) also increased CHAP activityin plate lysis by a factor of 64.

In order to determine the specificity of the HydH5 and its derivativefusions, the plate lysis assay was performed using cells from differentspecies and genera grown at OD_(600 nm)=0.5. The different bacterialcells suspensions were exposed to 2 μM protein solutions in the platelysis assay. We found that 10 μl of 2 μM HydH5-derived fusions,HydH5-SH3b and HydH5CHAP-SH3b, were able to lyse all S. aureus and S.epidermidis strains (14 tested; Table 1). In contrast, 10 μl of 2 μMHydH5-Lysostaphin was able to lyse all the S. aureus strains and only 2out of 4 S. epidermidis strains. S. epidermidis lysis could be achievedat 4 μM (data not shown). Other bacteria belonging genera Bacillus,Streptococcus, Clostridium, Lactococcus, Leuconostoc, Lactobacillus,Listeria and Enterococcus were not affected by any of the staphylolyticproteins. The lytic activity of HydH5 and its derivative fusions isspecific for staphylococci. It was observed that the degree of lyticactivity on S. epidermidis strains was generally lower than thatobserved on S. aureus group. Moreover, a different spectrum of lyticactivity was noted for each protein. HydH5 and HydH5-Lysostaphin seem tobe the less active proteins in this assay. HydH5CHAP-SH3b showed thehigher lytic activity even against S. epidermidis strains (Table 1). Inaddition to the differences between species, strain origin also seems toplay a role in determining the lytic spectrum since HydH5-SH3b andHydH5-Lysostaphin were more active against bovine strains than clinicalstrains. This difference is more pronounced in the case ofHydH5-Lysostaphin (Table 1).

TABLE 1 Lytic spectrum of HydH5 and its derivative fusions. Bacterialstrains were exposed to 2 μM of each protein. PROTEIN Lyso- STRAIN HydH5HydH5Lyso HydH5SH3b CHAPSH3b staphin S. aureus: Bovine strains Sa9 +* +++++ +++ +++ Sa6 − +++ +++ +++ +++ Sa11 + +++ +++ +++ +++ AFG1 + ++ ++++++ +++ AC9 + + +++ +++ +++ S. aureus: Human strains N315 − ++ +++ ++++++ 96 − + ++ +++ +++ 143 − + + ++ +++ 445 − ++ +++ +++ +++ c4 + ++ ++++++ +++ S. epider- midis B1CD2 + − +++ +++ + C213 − ++ +++ +++ ++Z2LDC17 + + ++ +++ − SILDC3 − − ++ +++ ++ *Strong lytic halo (+++),medium (++), weak (+) and no halo (−) was indicated.

Proteins were also tested by turbidity reduction assay (FIG. 2C)performed against live S. aureus Sa9 cells prepared as previouslydescribed (Donovan and Foster-Frey, supra; Becker et al. 2009, supra).The turbidity assay measures the drop in optical density (OD) resultingfrom lysis of the target bacteria with the HydH5-derived protein. Astandardized turbidity assay modified from (Donovan et al. 2006a. Appl.Environ. Microbiol. 72:2988-2996) with S. aureus grown to logarithmicphase (Δ_(600 nm)=0.4-0.6) at 37° C. in TSB (Tryptic Soy Broth, DIFCO,Franklin Lakes, N.J.) were performed in a 96 well dish and analyzed in aplate reader as described previously (Becker et al. 2009b., supra). Logphase cultures were harvested at 4° C. by centrifugation and stored onice less than 4 hours until just before the assay when they weresuspended in 150 mM NaCl, 10 mM Tris-CI, pH 7.5 or 50 mM Phosphatebuffer, pH 7.5 to an Δ_(600 nm) ˜1.0. Enzyme samples are added to threewells of a 96 well dish in 100 μl of buffer (Ni-NTA elution buffer orstorage buffer were shown to be equivalent). All samples are performedin triplicate. The assay is started by the addition of 100 μl of cellsin buffer at Δ_(600 nm) ˜1.0 via multichannel pipettor. A ‘no enzymecontrol’ of buffer and cells is included. Δ_(600 nm) readings are takenevery 20 seconds for 5 minutes. The readings for each well aretransferred electronically to an Excel spreadsheet where they areanalyzed in a sliding window over each group of 3 consecutive timepoints during the five minute period, to identify the highestinstantaneous change in Δ_(600 nm) for each well. The absolute values ofΔA_(600 nm) for each group of 3 time points are ranked and the optimumchosen based on highest absolute value and reproducibility in thetriplicate wells. A similarly calculated buffer plus cells alone controlvalue from triplicate wells is then subtracted from the highest rankedvalue for each experimental well, and the values for the triplicatewells averaged to give a ΔOD_(600 nm)/minute. This value is then dividedby the concentration (μM) of enzyme protein in the sample tested to givea specific activity ΔOD_(600 nm)/μM/min. The turbidity reduction assaysare repeated with multiple independent protein isolations to verify theresults, but only representative assays are presented, due to the highday-to-day variability in the results, presumably due to variations inthe cell culture preparations.

This assay was performed using 1 μM of purified proteins. Specificactivity of the proteins was expressed as ΔOD_(600 nm) min⁻¹ μM⁻¹. Thefull-length recombinant HydH5, synthesized from the E. coli codonoptimized version of orf58opt, showed a lytic activity in this assay incontrast to that previously observed in HydH5 from the standard orf58(Rodriguez et al. 2011, supra). The recombinant HydH5 was able to lyselive S. aureus Sa9 cells and had a specific activity of 0.033±0.009ΔOD_(600 nm) min⁻¹ μM⁻¹ (FIG. 2C). Similar results were obtained forHydH5CHAP domain but about 1.4-fold reduction in specific activity(0.023±0.001 ΔOD_(600 nm) min⁻¹ μM⁻¹) was observed when compared tofull-length HydH5 activity. Fusion proteins, HydH5-Lysostaphin andHydH5-SH3b, showed a 2.5- and 1.7-fold higher specific activity than theparental protein HydH5, respectively. HydH5CHAP-SH3b showed an activity4.8-fold greater than the CHAP domain. The specific activity ofHydH5CHAP-SH3b was calculated to be 0.109±0.023 ΔOD_(600 nm) min⁻¹ μM⁻¹the highest lytic activity obtained from HydH5 fusions. Therefore,C-terminal lysostaphin fusions conferred an enhanced staphylolyticactivity to HydH5 and its catalytic domain CHAP.

Example 4 Synergy

To determine the interaction of HydH5 and its derivative fusions withthe endolysin LysH5 a standard checkerboard dilution assay was performedusing live S. aureus Sa9 cells in HydH5 activity buffer to a finalOD_(600 nm) of ˜0.8. Checkerboard tests were performed between LysH5(Obeso et al., supra) and HydH5 and between LysH5 (Acc. Number EU573240)and HydH5-derived constructs as described in Garcia et al. (2010,supra)). Initially, we determined the minimum lytic concentration (MLC)of each protein: HydH5 (56.7 μg/ml), HydH5-Lysostaphin (12.5 μg/ml),HydH5-SH3b (13.5 μg/ml), HydH5CHAP-SH3b (8.7 μg/ml) and LysH5 (2.5μg/ml). MLCs were defined as the lowest concentration at which anOD_(600 nm) less than 0.1 after 15 minutes of incubation at 37° C. wasobtained. Ranges of enzyme concentrations were thus: LysH5 (2.5 μg/ml to0.04 μg/ml); HydH5 (56.7 μg/ml to 0.06 μg/ml), HydH5-Lysostaphin (25.1μg/ml to 0.02 μg/ml), HydH5-SH3b (13.5 μg/ml to 0.01 μg/ml) andHydH5CHAP-SH3b (17.34 μg/ml to 0.03 μg/ml). The fractional inhibitoryconcentration (FIC) was calculated as the minimum lytic concentration(MLC) of the antimicrobial in combination divided by the MLC of theantimicrobial acting alone. Strong synergy exists if the sum of the twoFICs [ΣFIC=FICA+FICB] is <0.5 (Hall et al. 1983. J. Antimicrob.Chemother. 11(5):427-433). All the experiments were performed induplicate.

When HydH5 or its derived fusions were combined with LysH5, asynergistic effect was observed in all combinations. In the presence ofsubinhibitory concentrations of the HydH5-derived proteins, a lowerendolysin LysH5 concentration was needed to fully decrease the S. aureusSa9 OD_(600 nm). Representative graphs of the synergistic interactionbetween LysH5 and HydH5 and between LysH5 and each HydH5-lysostaphinconstruct are shown in FIG. 3. HydH5 and its fusions act synergisticallywith LysH5 against S. aureus. From the MLC checkerboard test, theaverage ΣFIC values for the four protein combinations were calculated(FIG. 3). All these values are indicative of strong synergisticinteraction and it could be concluded that the increased lysis observedwith these mixtures would also be more effective to inhibit S. aureusbacterial growth.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

The foregoing description and certain representative embodiments anddetails of the invention have been presented for purposes ofillustration and description of the invention. It is not intended to beexhaustive or to limit the invention to the precise forms disclosed. Itwill be apparent to practitioners skilled in this art that modificationsand variations may be made therein without departing from the scope ofthe invention.

1-18. (canceled) 19: An isolated antimicrobial fusion peptidoglycanhydrolase protein comprising either a non-truncated HydH5 peptidoglycanhydrolase or a functioning HydH5 CHAP domain from truncated HydH5 incombination with either lysostaphin or the SH3b cell wall-binding (CWB)domain of native lysostaphin. 20: The protein of claim 19 wherein saidfusion protein is HydH5 peptidoglycan hydrolase-lysostaphin, HydH5peptidoglycan hydrolase-SH3b, or HydH5CHAP-SH3b. 21: The protein ofclaim 20 wherein said protein is identified by SEQ ID NO: 10, SEQ ID NO:12, and SEQ ID NO: 14, respectively. 22: A composition useful for thetreatment of disease caused by the Staphylococcus strains for which thefusion proteins of the invention are specific and effective, whereinsaid composition comprises any one of the proteins of claim 20 and apharmaceutically acceptable carrier. 23: A composition useful for thetreatment of a disease caused by multidrug-resistant staphylococcalstrains including methicillin-resistant S. aureus (MRSA), wherein saidcomposition comprises any one of the fusion proteins of claim 20 and apharmaceutically acceptable carrier. 24: A antimicrobialstaphylococcal-specific composition comprising an isolated orrecombinant LysH5 endolysin together with HydH5 peptidoglycan hydrolaseor with an HydH5-derived fusion protein selected from HydH5peptidoglycan hydrolase-lysostaphin, HydH5 peptidoglycan hydrolase-SH3b,or HydH5CHAP-SH3b wherein said composition is functional for treatmentof diseases and infections caused by Staphylococcus strains where thecomposition comprises LysH5 and a particular HydH5 fusion protein eachin amounts that are ineffective alone but act synergistically togetherto effectively treat said diseases and infections. 25: A compositionuseful for the treatment of a disease caused by multidrug-resistantstaphylococcal strains including methicillin-resistant S. aureus (MRSA),wherein said composition comprises any one of the fusion proteins ofclaim 24 and a pharmaceutically acceptable carrier. 26: A compositionuseful for the treatment of disease comprising the composition of claim20 in combination with another sequence, or sequences, encoding one ormore disease-resistance properties. 27: A method of treating infectionand disease caused by staphylococci in an individual comprising:administering to said individual an effective dosage of a composition ofclaim 20, wherein said composition comprises an isolated recombinantpeptidoglycan hydrolase fusion protein having specificity and exolyticactivity for the peptidoglycan cell wall of untreated staphylococci andwherein said administration is effective for the treatment of diseasesand infections caused by Staphylococcus strains includingmultidrug-resistant staphylococcal strains and methicillin-resistant S.aureus (MRSA). 28: A method of treating infection and disease caused bystaphylococci in an individual comprising: administering to saidindividual an effective dosage of a composition of claim 24, whereinsaid composition comprises LysH5 and an isolated recombinantpeptidoglycan hydrolase fusion protein having specificity and exolyticactivity for the peptidoglycan cell wall of untreated staphylococci,each in amounts that are ineffective alone but act synergisticallytogether to effectively treat said disease and infection caused byStaphylococcus strains including multidrug-resistant staphylococcalstrains and methicillin-resistant S. aureus (MRSA). 29: A method oftreating mastitis in an animal comprising: administering to said animalin need of treatment for mastitis an effective dosage of a compositionof claim 20, wherein said composition comprises an isolated recombinantpeptidoglycan hydrolase fusion protein having specificity and exolyticactivity for the peptidoglycan cell wall of untreated staphylococci andwherein said administration is effective for the treatment of andreduction of severity of mastitis caused by Staphylococcus strainsincluding multidrug-resistant staphylococcal strains andmethicillin-resistant S. aureus (MRSA). 30: A method of treatingmastitis in an animal comprising: administering to said animal in needof treatment for mastitis an effective dosage of a composition of claim24, wherein said composition comprises LysH5 and an isolated recombinantpeptidoglycan hydrolase fusion protein having specificity and exolyticactivity for the peptidoglycan cell wall of untreated staphylococci,each in amounts that are ineffective alone but act synergisticallytogether to effectively treat or reduce the severity of said mastitiscaused by Staphylococcus strains including multidrug-resistantstaphylococcal strains and methicillin-resistant S. aureus (MRSA). 31: Akit, comprising a composition of any one of claims 22-26.